Glutathione (L-α-glutamyl-L-cysteinyl-glycine; GSH) is a well-known tri-peptide thiol found in virtually all cells. Glutathione disulfide (GSSG) is the oxidized dimmer of glutathione. Glutathione functions to reduce the disulfide linkages of proteins and other molecules, as an antioxidant, and is an essential compound in the gamma-glutamyl cycle, which is thought to effect transport of amino acids through cell membranes. Glutathione is also known to act as a cofactor in a variety of enzymatic reactions, including the glyoxylase reaction, the cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate, and the formaldehyde dehydrogenase reaction.
Glutathione is synthesized by the method of Ozawa, Y. et al., Bull Chem, Soc. Jpn. 53: 2592 (1980). Alternatively, glutathione is isolate from yeast by the method fo Bloch, K. et al., Methods in Enzymology 3, 603 (1957). Glutahione disulfide (GSSG) is synthesized by the method of Bitny-Szlachto et al., Acta Biochim. Pol. 17, 175 (1970). Glutathione and its disulfide are also readily available commercially and inexpensive.
U.S. Pat. Nos. 6,013,632 and 6,107,281 to Jones et al., the disclosures of which are incorporated herein by reference in their entireties, disclose the effective use of glutathione, its derivatives, and pharmaceutically-acceptable salts of the same and of similar compositions specifically containing thiol, sulfide, disulfide and/or mercapten groups, including N-acetyl-L-cysteine, for the prophylaxis and treatment of influenza virus infections. Delivery is disclosed as being preferably carried out in the form of a lozenge, drinking solution, mouth rinse, or nasal spray for the purpose of coating the oral/nasal passages and mucous membranes of the subject Thiol-containing compounds, however, are notorious for their unpleasant odor and taste and so the use of thiol compounds such as glutathione in oral and nasal formulations has not been well received.
Glutathione and other thiol-containing compounds are most effective in the treatment and prophylaxis of influenza virus when administered in their reduced form, i.e., where thiol or sulfide (—SH) groups are present Furthermore, the sulfurous taste and smell of thiol compounds like glutathione increases with the amount of oxidation. However, such compounds are highly prone to oxidative degradation.
Degradation of reduced glutathione via oxidation is caused by many factors, the most significant of which are exposure to high temperature and water. It is well known by those skilled in the analytical arts that standard analytical solutions of pure glutathione in water solution must be prepared fresh every 4 hours due to the loss of concentration of reduced glutathione through oxidation. High temperature exposure causes oxidative degradation of glutathione even faster than exposure to water, and the higher the temperature the faster the thiol compound oxidizes. Long-time exposure to water at a relatively low temperature will generate similar levels of oxidation as exposure to high temperature for a short time.
This fact prevents the easy incorporation of thiol compounds into oral or nasal vehicles in highly reduced form. Glutathione cannot be added to a candy product vehicle during production in a water solution do to its instability in water. Further, such vehicles generally have an aqueous component themselves and are prepared using heat, thus accelerating the oxidation of the thiol-containing compounds incorporated therein. The thiol compounds tend to become oxidized during the product preparation phase and are thus not available in the final product to the patient in their more effective, highly reduced form. For example, glutathione tends to be converted to glutathione disulfide when being incorporated into a candy-base, i.e. a lozenge or cough drop, as the glutathione must be added while the aqueous candy base is in a hot, molten form.
Accordingly, there is therefore a need in the art for methods of preparing compositions containing thiol-containing compounds having a reduced sulfur odor and/or sulfur flavor, as well as compositions prepared by such methods, and there is a further need for methods of preparing compositions including thiol-containing compounds wherein the thiol-containing compounds are protected from degradation.